Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. We addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, over-expression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3, and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 over-expression and were not seen with a V-1 mutant that cannot bind CP (FBM V-1). V-1 is present in molar excess over CP suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells over-expressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1 null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1 but not by FBM V-1. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, obtained evidence that V-1s ability to sequester CP is regulated by phosphorylation, suggesting cells may manipulate the level of active CP to tune their actin phenotype. CARMILs (Capping protein Arp2/3 Myosin I Linker) are 1000 residue, multi-domain scaffold proteins expressed from protozoa to man that have been studied extensively with regard to their ability to bind Capping Protein (CP) and reduce its affinity for the actin filament barbed end. CARMIL proteins also appear to play important roles in signal transduction, as they exhibit genetic and physical interactions with the Rac GEF Trio, and T cells lacking CARMIL-2 exhibit a profound block in signaling downstream of CD28, the major co-receptor (along with the T cell receptor) for T cell signaling. In previous work we showed that Dictyostelium CARMIL binds CP, the Arp2/3 complex and myosin I (through its SH3 domain) and is required for actin dependent processes such as chemotaxis and macropinocytosis to be robust. We now describe initial studies of CARMIL-GAP, a second Dictyostelium CARMIL that contains, in addition to all the normal CARMIL domains (including the CP binding domain), a 130 residue insertion that, by homology, is a GTPase activating (GAP) domain for Rho-GTPases. Like CARMIL, CARMIL-GAP localizes to actin-rich structures (e.g. macropinocytic crowns) and is expressed in both vegetative and starved, developing cells. While CARMIL-GAP null cell lines created by homologous recombination are viable, they grow very slowly in suspension culture. Interestingly, full length CARMIL-GAP, as well a C-terminal fragment containing the GAP domain, are very poorly expressed in wild type cells. Conversely, both molecules are strongly expressed if their GAP domains contain a point mutation (R737A) that blocks GAP activity in all characterized GAP proteins. Together, these results suggest that the GAP domain in CARMIL-GAP is functional, that its over-expression is toxic to cells, and that at physiologic levels it probably plays a significant role in regulating the nucleotide state of one or more of the eight Rac GTPases expressed in Dictyostelium. Current studies are focused on determining the extent of this regulation, and how that in turn controls actin assembly in cells.